Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Reclassification of Eimeria pogonae Walden (2009) as Choleoeimeria pogonae comb. nov. (Apicomplexa: Eimeriidae).

The presented paper provides a reclassification of Eimeria pogonae from Pogona vitticeps into the correct genus Choleoeimeria. A description of exogenous and endogenous stages of biliary coccidium is given. Sporulation of the oocysts was endogenous. The mature oocysts contained four sporocysts each with two sporozoites. Oocysts were ellipsoidal in shape, with average length/width ratio 1.7 and measured 28.4 (SD1.5) × 16.8 (SD 1.5). The micropyle, residuum, and polar granules were absent from the sporulated oocysts. Ovoidal in shape, sporosysts without Steida bodies contained residuum and two elongated and boat-shaped sporozoites. The endogenous stages of the coccidia were located mainly in the epithelium of bile ducts; however, single-epithelium cells of the gallbladder were also infected.

ß-1,3-glucan, which can be targeted by drugs, forms a trabecular scaffold in the oocyst walls of Toxoplasma and Eimeria.

UNLABELLED: The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., ß-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, ß-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is ß-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) ß-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of ß-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, ß-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. IMPORTANCE: We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain ß-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, ß-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of ß-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.

Crystalloid body, refractile body and virus-like particles in Apicomplexa: what is in there?

The phylum of Apicomplexa comprises parasitic protozoa that share distinctive features such as the apical complex, the apicoplast, specialized cytoskeletal components and secretory organelles. Other unique cytoplasmic inclusions sharing similar features have been described in some representatives of Apicomplexa, although under different denominations. These are the crystalloid body, present for example in Cryptosporidium, Plasmodium and Cystoisospora; the refractile body in Eimeria and Lankesterella; and virus-like particles, also present in Eimeria and Cryptosporidium. Yet, the specific role of these cytoplasmic inclusions in the cell cycle of these protozoa is still unknown. Here, we discuss their morphology, possible inter-relatedness and speculate upon their function to bring these organelles back to the attention of the scientific community and promote new interest towards original research on these elusive structures.

Movable computer ruler (MCR): a new method for measuring the size of Toxoplasma gondii cysts, tachyzoites and other selected parasites.

A new method for measuring the size of parasites and other objects using optical microscopy was developed using a specifically designed movable computer ruler (MCR) derived from digital images of a stage micrometer. Subsequently, MCR can be superimposed on images of parasites to measure their size. MCR derived from the stage micrometer under a particular objective lens can be used to measure the size of an object acquired by the same lens/microscope/camera system. The conditions are fixed for every superimposed image including width, height, pixel number and density. The MCR was tested using selected parasites, and shown to be as accurate as the ocular micrometer disk, screw micrometer eyepiece and image analysis software. The lower technical complexity of the MCR method makes it applicable even in laboratories with limited resources.

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes.

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.

Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies.

Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed after immunization of mice with homogenates of Eimeria bovis first-generation merozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targets. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recognized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 180 kDa after electrophoresis under reducing conditions and to a series of components when separated without reduction. The epitope of mAb 8-23F9 was destroyed by treatment of the antigen with endoglycosidase H and removal of phosphocholine (PC) by phospholipase C. Since mAb 8-23F9 does not recognize cytidine-linked PC, the data suggest that PC in combination with N-linked sugars and/or N-glycans is part of its epitope. In the case of mAb 9-21G9, endoglycosidase H did not alter the epitope. When E. bovis merozoite antigen was treated with phospholipase C the number of mAb 9-21G9-reactive constituents increased, suggesting that PC may otherwise mask the epitope. mAb 8-23F9 also bound to the apical area and the surface of E. bovis sporozoites and recognized a >200-kDa sporozoite component. When sporozoites invaded Vero cells in vitro, epitope-bearing components were released onto the host cell surface and became part of the early parasitophorous vacuole wall. At day 5 the binding of the mAb was again confined to the intracellular parasite. mAb 9-21G9 did not react with sporozoites but recognized the apical area of intra-cellular trophozoites on day 5 after invasion of host cells in vitro. When testing was done against a variety of other Apicomplexa in various assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gondii cystozoites.

Evaluation of the effect of peptidyl membrane-interactive molecules on avian coccidia.

This study examined the lytic effect of seven different synthetic peptidyl membrane-interactive molecules (Peptidyl-MIMs) on sporozoites of five different species of Eimeria infecting chickens and merozoites of two different species that infect chickens. All Peptidyl-MIMs (pMIMs) demonstrated antiparasitic effects at concentrations of 1-50 microM during incubation periods varying from 1 to 20 min. In addition, electron microscopy showed that ultrastructural degeneration of the pellicle of sporozoite stages of the parasites occurred within 5-10 min of exposure to 5-microM concentrations of three different pMIMs. Pore-like openings were seen in the pellicle of the sporozoites at the ultrastructural level, which indicated that the pMIMs had the same mechanism of action on the parasites as that reported from studies done on bacteria. A reduction in lesion scores was seen in chickens treated orally with 10-, 50-, or 75-microM concentrations of two different proteolytic stabilized (methylated) pMIMs after challenge with three different species of avian coccidia in battery-cage trials. Collectively these data indicate that pMIMs may be useful in the control of coccidiosis in poultry.

Structure and function of the parasitophorous vacuole in Eimeria species.

The intracellular life-cycle of Eimeria are located in the host cell within a membrane-bound parasitophorous vacuole. The invasion process and the formation of the parasitophorous vacuole are mediated by characteristic organelles within the apical complex. During invasion, the parasitophorous-vacuole membrane is manipulated by the parasite and functions later in the development cycle as a molecular sieve, allowing the exchange of metabolites between parasite and host cell. Unlike the cyst-forming coccidia, there is little evidence of parasitophorous-vacuole membrane transformation in the later stages of the lifecycle of Eimeria species. Compared with the human pathogens Plasmodium and Toxoplasma, rather little is known about the parasitophorous vacuole and parasitophorous-vacuole membrane of animal pathogens of the genus Eimeria.

Ultrastructure of endogenous stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in experimentally infected goat

The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 10 5 occysts/kg. The secondary meronts developed above the nucleous first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nuclelous and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleus. The macrogametes contain a nucleus, wall-forming bodies of type I and II. The young oocysts present a wall containing two layers and a sporont.

Characterization of a chicken monoclonal antibody that recognizes the apical complex of Eimeria acervulina sporozoites and partially inhibits sporozoite invasion of CD8+ T lymphocytes in vitro.

A stable chicken hybridoma secreting a monoclonal antibody (mAb) that detects the apical complex of Eimeria acervulina sporozoites has been developed by fusing a thymidine kinase (TK)-deficient chicken myeloma with spleen cells from chickens immunized with sporozoite antigen. The mAb, designated as 6D-12-G10, recognized the apical complex of a sporozoite of 20-21 kDa molecular mass on western blots. Immunoelectron microscopic examination revealed that mAb 6D-12-G10 stained the conoid antigen. Furthermore, mAb 6D-12-G10 inhibited the invasion of sporozoites into CD8+ T cells in vitro. These results suggest that the conoid may play an important role in the recognition and invasion of host cells by Eimeria sporozoites.

Fine structure studies of microgametogenesis of Eimeria adenoeides (Eimeriidae, Sporozoa) infecting turkeys in Egypt.

The Ultrastructure of microgametogenesis of Eimeria adenoeides was studied in the intestinal epithelium of experimentally infected turkeys' Meleagris gallopavo gallopavo. Microgamonts were recognizable by the presence of peripherally arranged nuclei and the presence of two centrioles between each nucleus and the limiting membrane of the gamont. A nuclear spindle apparatus and an intranuclear centrocone directed toward the centriole were observed. Each young microgamont was surrounded by a very narrow parasitophorous vacuole which widened during development and contained a few intravacuolar folds. Differentiation of the microgamete began when elevations of the limiting membrane appeared above the centrioles. This event was accompanied by the segregation of nuclear content into a dense osmiophilic portion and an electron-pale portion. A gradual protrusion of the dense portion of the nucleus and developing flagella into the parasitophorous vacuole was proceeded. Microgametes had an anterior perforatorium, a dense elongate nucleus, with an anteriorly positioned mitochondrion in a small groove of the nucleus. Usually two flagella could be detected per each mature microgamete.

Effect of monensin on ultrastructure and cellular invasion by the turkey coccidia Eimeria adenoeides and Eimeria meleagrimitis.

Freshly excysted sporozoites (SZ) of the turkey coccidia Eimeria meleagrimitis and Eimeria adenoeides were incubated at 41 C in concentrations of monensin from .01 to 1.0 microgram/mL, washed free of the drug, and either processed for phase, fluorescence, and transmission electron microscopy or inoculated into cultures of turkey kidney cells. Phase microscopy indicated that after 1.5 h incubation in 1.0 micrograms/mL monensin, about 60% of the SZ of E. meleagrimitis had become notably rounded or displayed localized protrusions. These alterations were accompanied by ultrastructural abnormalities (in 90% of the SZ) including vacuoles in the cytoplasm, bulging and separation of plasma membrane layers, and dense bands in the refractile bodies that extended toward the periphery of the refractile body. Similar morphological and ultrastructural changes were observed in over half of the E. adenoeides SZ after 2 h incubation in 1.0 micrograms/mL monensin. Additionally, some specimens contained a pycnotic nucleus that was usually surrounded by a large vacuole. After 4 h incubation, almost all of the SZ displayed some degree of ultrastructural damage. Indirect fluorescent antibody labeling with parasite-specific monoclonal antibodies demonstrated clouds of antigen surrounding the monensin-treated but not the untreated SZ, suggesting an increase in permeability with incubation in monensin. With both E. meleagrimitis and E. adenoeides, the structural changes were reflected in a significant inhibition of cellular invasion. The inhibitory activity of monensin was concentration- and time-dependent in that the greatest inhibition of invasion was observed in SZ incubated for 4 h in 1.0 micrograms/mL of monensin; shorter incubation times or lower concentrations of monensin having less effect.

The ultrastructural effect of amprolium medication on development of Eimeria adenoeides in turkeys.

The ultrastructural appearance of first-generation schizonts of Eimeria adenoeides was markedly altered in turkey poults fed 0.0125% amprolium-medicated feed. When compared with development seen in unmedicated control birds 48-72 hr postinoculation (PI), most of the schizonts present in the medicated birds were fragmented, contained enlarged nuclei, had swollen endoplasmic reticulum and Golgi apparatus, and showed no budding of merozoites. Other schizonts were almost completely degenerated and contained pyknotic nuclei, dense cytoplasm, and large intracytoplasmic vacuoles containing membrane whorls or lipid droplets. A few mature schizonts were seen in the medicated poults, and these did not appear to differ ultrastructurally from those seen in unmedicated control birds. Ultrastructurally normal second-generation schizonts and the sexual stages were also seen in small numbers in the medicated turkey poults 72-120 hr PI. Mature sexual stages were seen much earlier in medicated turkeys--at 96 hr PI--than in the unmedicated poults.

Isolation and partial characterization of rhoptries and micronemes from Eimeria nieschulzi zoites (Sporozoa, Coccidia).

Homogenization and subcellular fractionation of sporozoites of Eimeria nieschulzi have allowed the recovery of highly enriched fractions of rhoptries and micronemes. The isolated organelles kept their in situ morphological characteristics. Their apparent densities in sucrose solutions were approximately 1.18 g/cm3 for rhoptries and 1.14 g/cm3 for micronemes. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the microneme fraction showed two major polypeptides at 220 and 94 kDa. The rhoptry fraction contained at least three predominant peptides at 200, 150, and 63 kDa. Micronemes were also isolated from third-generation merozoites of the same species; their characteristics were identical to those of the organelles isolated from sporozoites.

Scanning electron microscopy of damage to the cecal mucosa of turkeys infected with Eimeria adenoides.

White Wrolstad turkeys were each inoculated with 100,000 Eimeria adenoides oocysts and killed on days 4-14 postinoculation. Tissue samples, obtained from 4 areas of the ceca comparable to areas examined in chickens infected with E. tenella in previous studies, were processed by a modification of the osmium-thiocarbo-hydrazide-osmium technique and examined with a scanning electron microscope. The pathologic situation found in turkeys was slightly different from that in the ceca of chickens infected with E. tenella. The mucosal lesions are most severe at the proximal end of an infected cecum. Surface disruption was far less severe than with cecal coccidiosis in chickens of the same age exposed to an equal number of infective oocysts. Rupture of the epithelial cell often caused the mucosal surface to present a honeycomb appearance. Some specific stages of the life cycle were identified, including schizonts and oocysts.

The effects of heat and cobalt-60 gamma-radiation on excystation of the rat coccidium, Eimeria nieschulzi Dieben, 1924.

Sporulated oocysts of Eimeria nieschulzi Dieben, a rat coccidium, were exposed for 1 hr to Cobalt-60 gamma-radiation (15, 30, or 60k-rads), to heat (35, 40, or 45 C) , or to both concurrently (15, 30, OR 60 K-RADS AT 35 C) to compared the excystation capabilities of treated vs nontreated parasites. Intact treated oocysts appeared structurally unaltered when viewed with the light microscope. Excystation of sporozoites occured in all treated groups when their sporocysts were exposed to a trypsin-sodium taurocholate (TST) fluid, but after 150 min in TST the excystation rate was significantly lower than in nontreated sporocysts. Sporozoites which excysted from treated sporocysts were abnormal both in the excystation process and in their form and movement once outside the sporocysts.

[Light and electron microscope studies on two types of giant schizonts (globidial schizonts) in the small intestine of sheep (author's transl)]. / Lickt- und elektronenmikroskopische Untersuchungen an zwei Typen von Riesenschizonten aus dem Dünndarm des Schafes

The jujunum of sheep was investigated for globidial schizont infection. Two types of giant schizonts, which had a maximum diameter of about 500 mum, were observed. Each type was found to contain morphologically different parasites. In type 1 the parasites were hook-shaped with a length of about 15-18 mum. Often they had an additional tight lacing at the apcial pole. The merozoites of the second type were spindle-shaped, measuring only 9-12 mum in length. Both types of parasites had 22 subpellicular microtubles beneath their pellicle. The parasites observed here differed clearly from those of globidial schizonts from the abomasum as well as from those of sarcosporidian cysts of the same host. In light microscopy the globidial schizonts from the jejunum of sheep seemed to be covered by a thin, mono-layered wall. By means of electron microscopy, it was shown, that this layer is identical with the host-cell cytoplasm enclosing utricle-like a giant, membrane-bound parasitophorus vacuole, which in both types of schizonts was closely filled with parasites and an enclosing ground substance. The host cell of the second type of schizonts had always numerous microvilli at the outer surface. The inner surface of the host cell formed numerous intravacuolar tubules of about 50 nm in diameter and with a considerable length. The cytoplasm of the host cells appeared always very condensed. Summarizing it was shown that the globidial schizonts from the jejunum differed clearly from those in the abomasum as well as from the sarcosporidian cysts. Thus it can be stated that more species than supposed up to now form giant schizonts within the digestive system of sheep causing the lesions of high economical importance.

Ultrastructural studies on the endogenous development of Eimeria brunetti.

The schizonts of Eimeria brunetti were studied in the epithelial cells of the small intestine of infected chickens. The morphology of the host/parasite relationship was typical of that reported for other Eimeria spp. The initial dedifferentiation of the infecting organism generally occurred within the parasite cytoplasm but it was also observed that cytoplasm containing organelles could be budded off into the parasitophorous vacuole. Development into the schizont was accompanied by cytoplasmic growth and nuclear division. During nuclear division an eccentrically located nuclear spindle was present. Merozoite formation was initiated just below the limiting membrane of the schizont and was associated with the final nuclear division. The merozoites developed as protrusions from the schizont surface and merozoite organelles developed within these cytoplasmic projections. From this early stage, the developing merozoite grew and matured and the fully formed merozoites were found attached to a small residual mass of schizont cytoplasm. The 1st generation schizont is the only one which can be separately characterized. It differs from those of subsequent generations in a) possessing a refractile body, b) being larger and producing a larger number of merozoites, c) possessing invaginations of the limiting membrane, and d) the intra vacuolar folds of its parasitophorous vacuole are more extensive.

Sporozoites of Eimeria acervulina within intestinal macrophages in normal experimental infections: an ultrastructural study.

Sporozoites of Eimeria acervulina were observed in macrophages of the intestinal epithelium 5 and 6 days post-infection. These sporozoites lay within a well developed parasitophorous vacuole, were normal in structure and showed no signs of development. Macrophages harbouring sporozoites showed considerable structural changes, most pronounced being an absence of lysosomes, an enlarged nucleolus and extensive proliferation of the Golgi complex and endoplasmic reticulum. Possible mechanisms of survival and transport of sporozoites to preferred sites of development are discussed.